Research And Application On The Quantitative Detection Technology Of Vibrio Parahaemolyticus

Vibrio parahaemolyticus is halotolerant bacteria,which mainly exists in various sea water areas,seabed growth,sludge and fish,shrimp and shellfish seafood.This pathogenic bacterium can be mostly spreading through cross contamination of fresh fish,shrimp,shellfish,incompletely cooked seafood or uncoupled raw and cooked seafood,which may cause food-borne diarrhoeal illness.For a long time,V.parahaemolyticus has been detected by qualitative method,most of them are determined by traditional bacteriological MPN method or plate method.In this study,MPN-PCR,PMA-PCR and digital PCR were used to detect the virulence factors of Vibrio parahaemolyticus in raw oysters and clams in Xiamen.Tlh,tdh and ORF8 genes of Vibrio parahaemolyticus were detected by MPN-PCR.The MPN values of tlh,tdh and ORF8 genes were 3?>1100mpn/g,while trh was slightly lower,which was 3-43MPN/g.In the clam,tlh was positive,of which MPN value was 7.2?>1100 MPN/g,trh detection rate was the lowest,to 3-11 MPN/g.Tdh gene was less detected in raw aquatic products and environmental samples,tlh was more common in natural V.parahaemolyticus.Vibrio parahaemolyticus with tlh gene could also be detected in the environment and clinical samples,the whole detection process included enrichment culture,samples treatment,and PCR amplification,for those the total time was less than 24 hours,and the rapid quantitative detection of virulence factors for V.parahaemolyticus was preliminarily realized.In this study,the tox R gene with good specificity was used as the detected target gene,and a quantitative method of fluorescence PCR was established.It was found that adding 10 mg/L PMA to the suspension of the sample,could effectively inhibit the amplification of DNA in the dead bacteria(2×10~6 CFU/m L),while when the concentration of PMA was less than or equal to 20 mg/L,there was no significant effect on the amplification of Vibrio parahaemolyticus;at the same time,adding PMA to the mixture of dead and live bacteria,with the increase of the proportion of live bacteria.PCR showed a significant inhibition when compared to the positive control(P<0.05).It suggested that this method could reduce the detection time of pathogenic bacteria from 5-6 d to 3 h.The sensitivity was 20 CFU/m L,and the specificity was up to 99%.In the study of digital PCR,the nucleic acid was extracted from 200L enrichment solution after culture,and then water in oil,micro drop,PCR and micro drop count were carried out.The whole detection process was no more than 72 h.The maximum positive copy number of virulence factor was 7500,and the minimum was as low as 4 copies.It could be inferred that the copy number of each unit in the original template was about 33750.There was no need for the standard sample in the relative quantitative analysis during the whole micro drop detection process,and dead and live bacteria in the sample could be detected directly.The above results showed that MPN-PCR.PMA-PCR and digital PCR could be used to determine the total amount,virulence factors and the numbers of dead and live bacteria of Vibrio parahaemolyticus in seafood,which might provide a reference for quantitative detection of the pollution of this bacterium.